Process of determining the efficacy of drug treatment in HIV infected subjects

ABSTRACT

The present invention provides a process for determining the efficacy of anti-viral therapy in an HIV-infected subject receiving such therapy. The process includes the steps of a) detecting the level of transcriptionally active HIV in monocytes of the subject at a plurality of different times, b) comparing the detected HIV levels, and c) correlating changes in the detected HIV levels over time with the therapy. The process can be used to monitor the efficacy of treatment with any anti-HIV agent such as AZT, 3TC, DDC, Indivar, or Saquinavir. Decreases in HIV levels over time indicate an efficacious treatment. Increases in detected HIV levels over time indicate resistance to treatment.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is the national stage application ofinternational application PCT/US97/14870, filed Aug. 22, 1997, whichdesignates the United States, a continuation in part of U.S. ProvisionalPatent Application Ser. No. 60/024,404, filed Aug. 22, 1996 and acontinuation-in-part of U.S. Provisional Patent Application Ser. No.60/028,169, filed Oct. 11, 1996.

TECHNICAL FIELD OF THE INVENTION

The field of the present invention is HIV therapy. More particularly,the present invention pertains to a process of determining the efficacyor efficiency of drug treatment in HIV infected subjects by determiningthe level of HIV in monocytes of the subject.

BACKGROUND OF THE INVENTION

HIV is known to productively infect a variety of different cell types invitro and in vivo. The extent to which HIV infects and replicates inthese cells has important implications concerning dissemination fromportals of entry, cell function, and disease progression given thefinite number of target cells. End products of viral replicationincluding expression of unspliced HIV mRNA and plasma free virus has ledto virologic determinants as a measure of disease state and therapeuticefficacy. A marked increase in the ratio of unspliced to spliced HIVmRNA as might occur during the shift from latent to productive infectionprecedes precipitous drops in CD4 count. Plasma viral load has beenshown to correlate with disease progression and has been used todetermine HIV kinetics in viva. These measurements, however, fail toprovide information on the cell type of origin-a weakness consideringthe effects of HIV expression on cell function, the role of infectedcells in transmission and dissemination, and the therapeutic potentialof blocking cell type specific co-receptors.

Plasma viral burden analysis have allowed researchers to estimatekinetic parameters of HIV-1 life cycle in vivo. The life span ofproductively infected T-lymphocytes was estimated to be 2.2 days. Thedetection and quantification of productively infected lymphocytes,therefore, is technically difficult requiring very sensitive techniques.Further, the turnover of these cells may be too rapid to measure thesecells on a continuous basis. In addition, the contribution of free virusto the infective pool by cell types not destroyed by viral replicationhas not been experimentally addressed and has only been included as anaside in most kinetic models.

The transmission of HIV characteristically involves the early appearanceof NSI (non-syncitial inducing), macrophage tropic viral isolatesdespite the presence or absence of SI (syncitial inducing) and NSIvariants in the donor. Effective anti-HIV immune responses have beensuggested to temper the replication of SI variants after transmission.Following seroconversion, SI variants start to appear as a result ofimmune dysfunction. Some studies, however, suggest that NSI, macrophagetropic isolates persist throughout the disease course in spite of theabundance of SI variants.

Recent data lends support to the model that selective transmission mayoccur as the virus penetrates mucosal surfaces and encounters cells ofmonocyte/macrophage lineage. The virus can then be distributedthroughout the lymphoid system and tissue compartments via these cells.Once in the lymphoid compartment, the virus encounters the overwhelmingmajority of the body's lymphocytes. The virus accumulates in andeventually destroys the T-cell reservoir leaving only macrophages inT-cell depleted lymph nodes. Although free virus determinations havehelped define HIV-1 kinetics in vivo, resent elucidation of macrophageand T-cell tropic coreceptors demands further characterizations of thecells producing virus at various times during disease progression andduring antiretroviral therapy.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a process of determining or monitoringthe efficacy of drug treatment in HIV infected subjects. In accordancewith this process, the levels of HIV RNA in monocytes of the subject aremeasured over the course of treatment with one or more drugs (e.g., AZT,3TC, DDC). As drug treatment efficacy increases, the levels of HIV RNAin monocytes decreases. Conversely, where a subject develops resistanceto a drug, that resistance is evident from an increase or lack ofdecrease in monocyte HIV RNA levels. In other words there is a directcorrelation between the effectiveness of treatment and monocyte HIV RNAlevels. Preferred monocytes for use in this invention are CD14⁺monocytes.

In accordance with the present invention, dual immunophenotyping PCR insitu hybridization (DIPDISH) is used to detect cells containing HIV-1DNA, dual immunophenotyping fluorescence in situ hybridization (DIPFISH)is used to detect and quantify gag-pol mRNA in cells and quantitativeRNA analysis is used to quantify plasma viral load. The presentinvention discloses that monocytes, and particularly CD14⁺ monocytes,are persistently productive of HIV message. Furthermore, the levels ofHIV mRNA in those monocytes respond in parallel with plasma viral loadto drug therapy. As viral message production is an earlier event invirion production a process of the present invention is more a moresensitive indicator of drug efficacy and drug resistance than prior artmethods.

Productively infected cell types in patients infected by HIV have beenidentified and quantified. As shown previously, very few CD4 positivelymphocytes were productively infected by HIV although many containproviral DNA. The present invention discloses that monocytes are themajor productively infected cell type in HIV seropositive individualsand viral production in these cells is altered by antiretroviraltherapy. The percentage of productively infected monocytes correspondedwith viral burden analysis in patients on no, single, combination, andtriple drug therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, which form a portion of the specification:

FIG. 1 shows a time course experiment of ACH-2 cells stimulated withTNF-α and detected using FISH. Less than 5 percent of the cells expressany HIV-1 mRNA at time point 0 (A), expression of HIV-1 multiply splicedmRNA is produced within 1 hour (B) and unspliced transcripts start to beproduced at the 12 hour timepoint (C), and expression of all mRNA isseen in over 90% of the stimulated cells at 24 hours (D). The ACH-2 cellline (AIDS Research and Reagent Program, NIAID, NIH Bethesda, Md.),containing a single copy of integrated HIV-1 proviral DNA per cell, washarvested at an early passage number and used as the HIV-1 infected cellcopy number control cells were stimulated with μg/ml TNF-α.

FIG. 2a shows dot plots of patient PBMCs following DIPFISH. To determinethe cell types containing HIV RNA using DIPFISH cells are labeled withoptimized concentrations of phycoerythrin conjugated antibodies specificfor the cell types of interest (CD4, (FIG. 2a), CD14 (FIG. 2b)) andfixed and permeabilized by the addition of 50 μl of Permeafix (OrthoDiagnostics, Inc.) per 10⁶ cells at ambient incubation temperature forat least 60 minutes. The cells are then washed twice in PBS, pH 7.4 atambient temperature and once in 2×SSC at ambient temperature. The cellsare then resuspended with 50 μl hybridization buffer containing acocktail of 5- or 6-carboxyfluorescein-labeled oligonucleotides specificfor HIV RNA-Positive (ribosomal RNA) and negative control cocktails areused in replicate samples. Probe was hybridized to target for 30 minutesat 43° C. in a water bath. The cells were washed for 5 minutes withbuffer A at 42° C., 30 minutes with buffer B at 42° C. Autofluorescencewas quenched using quenching reagent. Multiparameter analysis of cellsurface molecules and HIV RNA was performed on a Coulter XL flowcytometer (Coulter, Hialeah, Fla.).

FIG. 2b shows a scattergram illustrating the relationship ofproductively infected monocytes (abscissa) and plasma viral load(ordinate) in patients on no ♦, single ▪, combination , and triple ×therapy. Forty HIV seropositive patients from the VA Lakeside Hospitalfollowed routinely were evaluated using CD4 count, DIPDISH, DIPFISH, andquantitative RNA. Five HIV seronegative patients were evaluated usingthe same assays. Patients were either on no therapy, single drug therapywith AZT (200 mg three times/day) or DDC (0.75 mg three times/day),combination therapy with AZT and 3TC (150 mg two times/day), or tripletherapy with AZT 3TC and either Indinavir (800 mg three times/day) orSaquinavir (600 mg three times/day). All patients were on therapy for atleast 30 days prior to blood donation. PBMCs were isolated from freshheparinized blood layered on a Histopaque 1077 (Sigma, St. Louis, Mo.)discontinuous density gradient and centrifuged at 600×g for 30 minutesat ambient temperature. The turbid layer was removed, washed twice with3 volumes of RPMI and once with phosphate buffered saline (PBS).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a process for determining the efficacy ofanti-viral therapy in an HIV-infected subject receiving such therapy.The process includes the steps of a) detecting the level oftranscriptionally active HIV in monocytes of the subject at a pluralityof different times, b) comparing the detected HIV levels, and c)correlating changes in the detected HIV levels over time with thetherapy. The process can be used to monitor the efficacy of treatmentwith any anti-HIV agent such as AZT, 3TC, DDc, Indivar, or Saquinavir.Decreases in HIV levels over time indicate an efficacious treatment.Increases in detected HIV levels over time indicate resistance totreatment.

The level of transcriptionally active HIV is detected by measuring thelevel of HIV mRNA and, preferably gag-pol HIV mRNA. The HIV mRNA isdetected using in situ hybridization. In situ hybridization isaccomplished by exposing monocytes in situ to an oligonucleotide probethat specifically hybridizes to at least of a portion of the HIV mRNA.The probe is labeled with a detectable marker, the detection of whichindicates the presence of HIV mRNA.

A process of this invention is particularly useful when the monocytesharboring the transcriptionally active HIV are CD14⁺ monocytes. CD14⁺monocytes are identified in blood samples of the subject usingimmunophenotyping. In accordance with such immunophenotyping, a bloodsample is exposed to an antibody directed against CD14⁺, which antibodyis labeled with a detectable marker. In a preferred embodiment, theimmunophenotyping and in situ hybridization are carried outsimultaneously. In accordance with that preferred embodiment, CD14⁺ islabeled with an anti-CD14⁺ antibody having a first detectable labelattached thereto, the labeled monocytes are then fixed and permeabilizedin the presence of an oligonucleotide probe that hydridizes to the HIVmRNA, which probe is labeled with a second detectable marker, and theamount of both detectable labels measured. Preferably, the first andsecond detectable labels are fluorescent labels. A preferred firstdetectable label is phycoerythrin. A preferred second detectable labelis 5- or 6-carboxyfluorescein.

The level of HIV RNA in monocytes is determined in blood samples fromthe subject using a combination of cellular antigen (e.g., CD14)detection (using labeled antibodies) and in situ hybridization using atagged probe directed against a region of HIV RNA. Details of suchmethods are set forth below. A detailed description of simultaneous insitu cellular antigen detection and nucleic acid amplification can befound in U.S. Pat. No. 5,843,640, filed Aug. 30, 1995, the disclosure ofwhich is incorporated herein by reference.

Study subjects

Thirty-seven HIV seropositive patients from the VA Lakeside Hospitalfollowed routinely were evaluated using CD4 count, DIPDISH, DIPFISH, andquantitative RNA analysis. Ten HIV seronegative patients were evaluatedusing the same assays. Patients were either on no therapy, single drugtherapy with AZT (200 mg, three times a day) or DDC (0.75 mg, threetimes a day), combination therapy with AZT and 3TC (150 mg, two times aday), or triple therapy with AZT 3TC and either Indinavir (800 mg, threetimes a day) or Saquinavir (600 mg, three times a day). All patientswere on therapy for at least 30 days prior to blood donation.

Histogram gates were set based on a negative control cocktail directedagainst cytomegalovirus and on a positive control cocktail directedagainst 28 ribosomal RNA. Fine tuning of the gates on each patientsample were set based on internal HIV-negative and CD4 or CD14 negativepopulations. CD4⁺ T-lymphocytes containing HIV-1 DNA ranged from 4% to43%. The percentage of cells containing HIV-1 DNA did not correlate withCD4 count or drug therapy suggesting that this population may be areservoir for defective viral genomes, nonproductive infection ofresting T-cells, or provirus capable of subsequent activation.

Cells and cell lines

PBMCs were isolated from fresh heparinized blood layered on a Histopaque1077 (Sigma, St. Louis, Mo.) discontinuous density gradient andcentrifuged at 600×g for 30 minutes at ambient temperature. The turbidlayer was removed, washed twice with 3 volumes of RPMI and once withphosphate buffered saline (PBS). The ACH-2 cell line (AIDS Research andReagent Program, NIAID, NIH, Bethesda, Md.), containing a single copy ofintegrated HIV-1 proviral DNA per cell, was harvested at an earlypassage number and used as the HIV-1-infected cell copy number control.

Dual Immunophenotyping/Fluorescence in situ Hybridization (DIPFISH).

To determine the cell types containing HIV RNA using DIPFISH, cells werelabeled with optimized concentrations of phycoerythrin (PE)-conjugatedantibodies specific for the cell types of interest (CD4, CD14) and fixedand permeabilized by the addition of 50 μl of a water-soluble,non-aldehyde fixative, Permeafix (Ortho Diagnostics, Inc.) per 10⁶ cellsat ambient incubation temperature for at least 60 min. The cells werethen washed twice in PBS, pH 7.4 at ambient temperature and once in2×SSC at ambient temperature.

The cells were then resuspended with 50 μl hybridization buffercontaining a cocktail of 5- or 6-carboxyfluorescein-labeledoligonucleotides specific for HIV RNA. Positive (ribosomal RNA) andnegative control cocktails were used in replicate samples. Probe washybridized to target for 30 minutes at 43° C. in a water bath. The cellswere washed with buffer at 42° C. Autofluorescence was quenched usingquenching reagent. Multiparameter analysis of cell surface molecules andHIV RNA was performed on a Coulter XL flow cytometer (Coulter, Hialeah,Fla.).

Dual Immunophenotyping/PCR-driven in situ hybridization (DIPDISH).

Cell samples were adjusted to a final concentration of 1×10⁶ cells/mi. A400 μl aliquot of each sample was centrifuged at 600×g for 2 minutes atambient temperature and the cell pellet was washed twice in PBS. Thecells were then fixed and permeabilized by the addition of 50 μl ofPermeafix (Ortho Diagnostics, Inc.) at ambient temperature for 60minutes. Cells were then pelleted as above, washed with PBS andresuspended in 190 μl of PCR reaction mixture consisting of 10 mM TrisHCL (pH 8.3); 50 mM KCL; 1.5 mM MgC₄; 0.25 mM each dATP, dCTP, dGTP;0.14 mM dTTP; 4.3 μM dUTP-11-digoxigenin; 100 pmole each forward andreverse primer; 1.0 μl (5 Units) Taq polymerase (Amplitaq, Perkin-Ehner,Norwalk Conn.); and gelatin 0.001% w/v.

The DNA in the reaction mixture was amplified in 500 μl tubes insertedinto the wells of a 48 well thermocycler (Perkin-Elmer Cetus) programmedfor 25 cycles of thermal denaturation (94° C., 1 minute), primerannealing (58° C., 2 minutes), and primer extension (74° C., 1.5minutes), with 5 seconds added for each of 25 cycles. Appropriatepositive and negative controls amplified with or without the addition ofTaq polymerase were simultaneously run with each sample. Afteramplification, cells were pelleted and resuspended in 25 μl of 10 mMTris HCL (pH 8.3), 50 mM KCL, and 1.5 mM MgC₄. A 100 mg aliquot of theappropriately labeled target specific oligonucleotide probe in 10 μg/mlsonicated herring sperm DNA (Sigma) was added to the reaction tube. Theproduct DNA was denatured at 95° C. for 3 minutes then allowed tohybridize with the respective oligonucleotide probe at 56° C. for 2hours.

After hybridization, the cells were washed for 30 minutes with 2×SSC/50%formamide/500 μg/ml bovine serum albumin (BSA) at 42° C., 30 minuteswith 1×SSC/50% formamide/500 μg/ml BSA at 42° C., 30 minutes with1×SSC/500 μg/ml BSA at ambient temperature and then briefly with PBS atambient temperature. Following the last wash, the cells were resuspendedin 80 μl of PBS and 20 μl strepavidin-phycoerythrin (PE) and incubatedfor 30 minutes at ambient temperature. The cells were then washed in PBSas described above.

Flow Cytometry.

The cell suspension was filtered through a 37 mm nylon mesh and analyzedby flow cytometry using an EPICS XL flow cytometer. Laser excitation was15 mW at 488 nm, and the FITC and PE florescence was detected withstandard optical filter set-up (550 dichroic, 525 bandpass (FITC) and585 bandpass (PE)). Instrument sensitivity was standardized before eachexperiment employing Immuno-Bright calibration beads (Coulter Source,Marriette, Ga.). The percent fluorescence-positive cells was determinedby integration over a range of 0.2% positive counts on the identicallytreated negative sample (100% uninfected PBMCs).

Viral Burden.

Quantitative RNA determinations were performed on plasma using theAmplicor RNA kit (Roche Molecular Systems, Alameda, Calif.) as permanufacturer instructions.

In peripheral blood, a significant proportion of PBMCs contain HIV-1 DNAwhile very few contain transcriptionally active virus. Simultaneousimmunophenotyping not only allowed unequivocal identification ofinfected cell subtypes but also enriched cell subtypes for the variousinfected cell types present in the blood. To determine the cell typeswith productive or latent HIV infection, immunophenotyping was combinedwith a novel in situ hybridization strategy using 220 5′- and 3′,6-carboxyfluorescein-labeled oligonucleotides complementary to gag-poltargets or with in situ PCR for proviral DNA. The in situ hybridizationexperiments were calibrated using the ACH-2 cell line with and withoutTNF-α stimulation. Time course experiments (FIG. 1) reveal that >98% ofunstimulated ACH-2 cells express high levels of HIV-1 mRNA which agreeswith previous estimates. Twenty four hours following stimulation, >90%of ACH-2 cells express high levels of HIV-1 mRNA which corresponds tobetween 300-400 genome copies per cell.

Using simultaneous immunophenotyping with CD4 and CD14 and in situhybridization, the data show that a significant proportion of monocytesharbor transcriptionally active HIV whereas very few CD4 T-lymphocytesharbor transcriptionally active virus at any point in time (FIGS. 2a and2 b). The percentage of productively infected monocytes ranged from <1%to 93% percent and the percentage of productively infected CD4lymphocytes ranged from <1 to 6% which, most likely, represents the morerapid turnover of productively infected CD4 cells and the persistence ofproductively infected monocytes. It was previously suggested thatproductive infection of monocytes occurred following differentiation tomacrophages and indeed a significant proportion of cells were found inperipheral blood with the CD14 low, CD16 high phenotype indicative ofmacrophage differentiation. Backgating on the cells expressing HIV RNA,however, did not reveal a specific, infected subset of CD14 positivecells.

To determine the relationship between the percentage of infected cellsand plasma viral load, plasma was obtained from the same sample used toisolate PBMCs and quantitative competitive RNA PCR was performed on eachsample (FIG. 3). Six groups were identified: those patients with highviral load, high percentage of infected monocytes; high viral load, lowpercentage of infected monocytes; low viral load, high percentage ofinfected monocytes; low viral load, low percentage of infectedmonocytes; and intermediate or low viral load, intermediate percentageof infected monocytes. A low viral load in the presence of a highpercentage of monocytes expressing HIV-1 mRNA may represent earlyindication of drug resistance. A high viral load in the presence of alow percentage of monocytes expressing HIV-1 mRNA may representproduction of free virus predominantly in other cell types as may occurin the phenotypic switch preceding symptomatic infection. Even in theextreme cases of a patient with a high viral titer in spite of tripletherapy or a patient with a low viral titer in the absence of therapy,the percentage of infected monocytes paralleled the viral load.

Nine out of fifteen patient (60.0%) with a high percentage of monocytesexpressing HIV-1 mRNA had viral loads over 50,000 copies/ml while fourout of twenty-three patient (17%) with less than 50% monocytesexpressing HIV-1 mRNA had viral loads over 50,000 copies/ml. As viralmessage production is an earlier event in the viral lifecycle, thismeasurement is likely a more sensitive indicator of drug efficacy anddrug resistance.

Productively infected cell types in blood from patients infected by HIVwere identified and quantified. CD14 positive monocytes were the majorpersistently productive cells infected with HIV. A specific subtype ofinfected monocytes was not identified even though CD14 low, CD16 highcells phenotypically resembling macrophages have been identified in theperipheral blood of HIV seropositive individuals. Interestingly,productively infected monocytes in several patients exhibited anincreased CD14 mean peak fluorescence relative to the HIV negativemonocytes in the same sample. Increases in CD14 expression in monocyteshas been shown in the peripheral blood of HIV-infected patients althoughthe presence of HIV in these cells was not determined in this previousstudy. CD14 is a glycosyl-phosphatidynositol-anchored molecule on thesurface of monocytes and to a lesser extent granulocytes. Monocyteactivation by lipopolysaccharide requires CD14, therefore alterations ofCD14 could conceivably explain the functional defect of monocytes at thecellular level in HIV-infected individuals. As shown previously, veryfew CD4 positive lymphocytes were productively infected by HIV althoughmany contain proviral DNA.

The transmission of HIV involves the early infection of antigenpresenting cells such as Langerhans cells and monocytes following breachof mucosal barriers. Virus is shuttled in these cells via peripheralblood to lymphoid and other tissue. Within lymphoid tissue free virusproduced by productively infected cells can infect other permissivecells or become trapped within the follicular dendritic cell (FDC)network.

Following infection, non-syncytium-inducing (NSI), macrophage tropicviral isolates predominate despite the presence or absence ofsyncytium-inducing (SI) and NSI variants in the donor. Effectiveanti-HIV immune responses have been suggested to temper the replicationof SI variants after transmission. Following progression to symptomaticdisease, SI variants start to appear as a result of immune dysfunction.The studies herein show that cells of the monocyte/macrophage lineageare likely a major reservoir of virus capable of producing virions. Yetthis reservoir has been omitted from mathematical models proposed toexamine the relationship between virion production and host celldestruction.

Much debate has centered on the question of whether monocytes areinfected, productively or latently, by HIV in vivo. The absolutemonocyte count and the absolute CD4 count were calculated in all of thepatients. Based on the percentage of productively infected CD4T-lymphocytes and productively infected monocytes, and given thatmonocytes release anywhere from 10% to 50% of the number of virions asT-cells, the amount of free virus contributed by these cell types shouldbe roughly equivalent. The exception to this would be patients with highviral loads and a low percentage of infected monocytes (FIG. 3). Thesepatients were all symptomatic with CD4 counts less than 200 suggestingthe switch to the T-cell tropic, SI phenotype had occurred in thesepatients. This may explain why other studies in patients with AIDSindicate that monocytes are rarely a source of virus.

Quantification of infected monocytes/macrophages is not only criticalfor a complete kinetic model of HIV infection but also for therapeuticmonitoring. Since these cells are not destroyed by viral production andpersistently infected cells such as macrophages, may have a lifespanfive to six times longer than infected T-lymphocytes, these cells areideal for monitoring HIV activity at the cellular level includinglymphoid tissue such as tonsil and lymph nodes.

Clearly, the contribution of productively-infected monocytes to the freevirus pool must be distinctly defined. With the discovery of new tropismdependent co-receptors for HIV and inevitable therapy directed atblocking these co-receptors, determination of productively infected celltypes and their contribution to the free virus pool is critical. Theease of the technologies used to define the viral lifecycle in thisstudy most likely portends the end of surrogate markers as we know themand reaffirms the trend toward viral lifecycle monitoring.

What is claimed is:
 1. A process for determining the efficacy ofanti-viral therapy in an HIV-infected subject receiving such therapy,the process comprising the steps of: a) detecting the level oftranscriptionally active HIV in monocytes of the subject at a pluralityof different times by simultaneously exposing the monocytes to anoligonucleotide probe that specifically binds to at least a portion ofHIV mRNA and exposing the monocytes to an antibody, wherein theoligonucleotide probe is labeled with a fluorescent label; b) comparingthe detected HIV levels; and c) correlating changes in HIV levels overtime with the therapy to determine the efficacy of the anti-viraltherapy is the subject.
 2. The process of claim 1 wherein the monocytesare CD14+ monocytes.
 3. The process of claim 2 wherein the CD14+monocytes are labeled with a antibody against CD14, which antibody islabeled with a detectable marker.
 4. The process of claim 3 wherein thedetectable marker is a fluorescent label.
 5. The process of claim 4wherein the detectable marker is phycoerythrin.
 6. The process of claim1 wherein the portion of HIV mRNA is the gag-pol portion.
 7. The processof claim 1 wherein the fluorescent label is a 5- or6-carboxyfluorescein.
 8. The process of claim 1 wherein the anti-viraltherapy is selected from the group consisting of treatment with AZT,3TC, Ddc, Indivar and Saquinavir.
 9. The process of claim 1 whereindecreases in HIV-RNA levels over time indicate an efficacious treatment.10. The process of claim 1 wherein increases in HIV-RNA levels over timeindicate resistance to treatment.